1. Field of the Invention
This invention relates to a biochemical reaction cassette equipped with a probe carrier such as a DNA micro-array that can suitably be utilized when examining the presence or absence of one or more than one genes originating from pathogenic microbes in a specimen such as a blood specimen to determine the health condition of a subject of medical examination. More particularly, the present invention relates to the structure of a biochemical reaction cassette for making the flow rate of the liquid flowing at least in a reaction chamber uniform.
2. Related Background Art
Many proposals have been made for methods utilizing a hybridization reaction by using a probe carrier which may typically be a DNA micro-array in order to quickly and accurately analyze the base sequence of a nucleic acid or detect a target nucleic acid from a nucleic acid specimen. A DNA micro-array is formed by rigidly and highly densely immobilizing a probe having a complementary base sequence relative to that of a target nucleic acid on a solid phase such as a bead or a glass plate. An operation of detecting a target nucleic acid using a DNA micro-array generally includes the following steps.
In the first step, the target nucleic acid is amplified by means of an amplification method, which may typically be the PCR method. More specifically, firstly, first and second primers are added to a nucleic acid specimen and a thermal cycle is applied to it. The first primer specifically binds to part of the target nucleic acid while the second primer specifically binds to part of a nucleic acid that is complementary to the target nucleic acid. As a double-stranded nucleic acid containing the target nucleic acid binds to the first and second primers, the double-stranded nucleic acid containing the target nucleic acid is amplified by way of an extension reaction. After the double-stranded nucleic acid that contains the target nucleic acid is amplified to a sufficient extent, a third primer is added to the nucleic acid specimen and a thermal cycle is applied to it. The third primer is labeled with an enzyme, a fluorescent substance, a luminescent substance or the like and specifically binds to part of the nucleic acid that is complementary to the target nucleic acid. As the third primer binds to the nucleic acid that is complementary to the target nucleic acid, the target nucleic acid that is labeled with an enzyme, a fluorescent substance, a luminescent substance, or the like is amplified by way of an extension reaction. As a result, a labeled target nucleic acid is generated when the nucleic acid specimen contains the target nucleic acid, where no labeled target nucleic acid is generated when the nucleic acid specimen does not contain the target nucleic acid.
In the second step, the nucleic acid specimen is brought into contact with a DNA micro-array to cause a hybridization reaction to take place between the specimen and the probe of the DNA micro array. The probe and the target nucleic acid form a hybrid when the target nucleic acid that is complementary to the probe is contained in the nucleic acid specimen.
In the third step, the target nucleic acid is detected. It is possible to detect if the probe and the target nucleic acid form a hybrid by means of the labeling substance of the target nucleic acid. Thus, it is possible to see the presence or absence of a specific bas sequence.
DNA micro-arrays that are adapted to utilize a hybridization reaction are expected to find applications in the field of medical diagnosis for identifying pathogenic microbes and that of gene diagnosis for examining the genetic constitution of a patient. However, the steps of amplification of nucleic acid, hybridization and detection are mostly individually conducted by using separate devices. Hence the overall operation is a complex one and it takes time for diagnosis. Particularly, when a hybridization reaction is conducted on a slide glass, the probe can become defective and/or contaminated when the slide glass is touched by a finger because the specimen immobilizing surface is exposed. Therefore, DNA micro-arrays need to be handled with utmost care. For the purpose of eliminating the above described problems, proposals have been made for the structure of a biochemical reaction cassette in which a reaction chamber is provided with a DNA micro-array so as to be able to conduct a hybridization reaction in the reaction chamber and also a subsequent operation of detecting a hybrid.
Japanese Patent Application Laid-Open No. H10-505410 discloses a structure for forming a cavity and a method of manufacturing such a cavity. Japanese Patent Application Laid-Open No. 2003-302399 and Japanese Patent Application Laid-Open No. 2004-093558 disclose chamber structures for preventing air bubbles from remaining in the initial liquid filling stages. Japanese Patent Application Laid-Open No. 2002-243748 discloses a structure for uniformly spreading liquid and forming a flow of such liquid.
With structures of biochemical reaction cassettes as disclosed in the above-cited patent documents, the volume of the reaction chamber is as small as tens of several μL and the height of the reaction chamber is also small to show a flatly extending profile. Such a structure provides an advantage of requiring only a small amount of reagent or some other liquid and producing a laminar flow in the reaction chamber. Additionally, the liquid in the reaction chamber may be agitated to efficiently give rise to a hybridization reaction of a probe and a target nucleic acid on a solid phase. The simplest way of agitating the liquid may be pushing and pulling the liquid at the injection port and rocking the liquid in the reaction chamber.
FIGS. 11, 12A and 12B of the accompanying drawings illustrate a biochemical reaction cassette as an example. The illustrated biochemical reaction cassette comprises a substrate 111 and a casing 112. Assume that liquid is filled in the reaction chamber 103 of the biochemical reaction cassette 110. If more liquid is fed from the inject port 106 thereof, the liquid flow rate 122 at and near the center of the reaction chamber 103 becomes higher than the liquid flow rates 121 and 123 at and near the opposite ends of the reaction chamber 103. Therefore, as the liquid in the inside is pushed and pulled at the injection port 106 or the discharge port 107 to rock the liquid in the reaction chamber 103, the frequency at which the probe on the solid phase contacts the target nucleic acid is differentiated between at and near the center of the reaction chamber 103 and at and near the opposite ends of the reaction chamber 103. Additionally, washing liquid is made to flow in the reaction chamber 103 after the end of a hybridization reaction in order to remove the nucleic acid that remains in the inside without reacting. At this time again, the rate at which the unreacted nucleic acid is removed and the probability at which the target nucleic acid that has reacted with the probe on the solid phase is pulled off are differentiated because of the difference of flow rate between at and near the center of the reaction chamber 103 and at and near the opposite ends of the reaction chamber 103. As a result, the luminance can vary at different positions on the probe at the time of detection to adversely affect the diagnosis.
With the arrangement of Japanese Patent Application Laid-Open No. H10-505410, while a laminar flow takes place in a cavity, the problem of difference of flow rate between at and near the center of the cavity and at and near the opposite ends of the cavity is not dissolved. With the arrangements of Japanese Patent Application Laid-Open No. 2003-302399 and No. 2004-093558, while liquid uniformly spreads in the chamber in the initial liquid filling stages, the flow rate of liquid in the chamber is not uniformized when the chamber is filled with liquid. Finally, with the arrangement of Japanese Patent Application Laid-Open No. 2002-243748, the structure is inevitably complex and hence the reduction of cost of manufacturing such a cassette is limited.